Glioblastoma multiforme (GBM) is a highly aggressive and deadly cancer that afflicts over 12,500 new patients in the United States every year. A landmark 2005 study demonstrated that the addition of temozolomide (TMZ) to standard-of-care therapy (surgical resection and radiation) offered a survival advantage, and since then surgery followed by TMZ+radiation has become the new standard-of-care treatment for GBM. TMZ acts by appending a methyl group to DNA, and if not repaired the methylation at the O6 position of guanine is lethal. The intratumoral expression of the DNA damage repair enzyme O6-methylguanine methyltransferase (MGMT) largely dictates patient response to TMZ: patients with MGMT-negative tumors have a 2-year survival of 49%, while those with MGMT-positive tumors have a 2- year survival of 15%. Over 60% of newly diagnosed GBM patients express high levels of MGMT, thus the majority of GBM patients do not benefit from TMZ therapy. As described in this proposal, we have devised novel compounds which, instead of appending a methyl group to DNA, append a functional group that should be difficulty/impossible for MGMT to remove. Thus these compounds should provide benefit to all GBM patients, regardless of MGMT status. In addition, prediction algorithms suggest these novel compounds should be more blood-brain barrier penetrant than TMZ. Recent developments in synthetic chemistry have now made the construction of these compounds possible, and in Specific Aim 1 the target compounds will be constructed and evaluated versus GBM cell lines in culture, and the MGMT- dependence of cell death will be assessed. Top compounds will be moved to Specific Aim 2, where they will be evaluated in sophisticated rat and mouse models of GBM. Our goal by the end of the two-year funding period is to have gathered appropriate preclinical data that will position these compounds for development toward a human clinical trial. This tightly-focused study that could provide a major breakthrough in GBM therapy is an ideal fit for the R21 funding mechanism.